Digital holography microscopy (DHM) is an optical technique which provides phase images yielding quantitative information about cell structure and cellular dynamics. Furthermore, the quantitative phase images allow the derivation of other parameters, including dry mass production, density, and spatial distribution. We have applied DHM to study the dry mass production rate and the dry mass surface density in wild-type and mutant fission yeast cells. Our study demonstrates the applicability of DHM as a tool for label-free quantitative analysis of the cell cycle and opens the possibility for its use in high-throughput screening.

We present first results on a method enabling mechanical scanning-free tomography with submicrometer axial resolution by multiple-wavelength digital holographic microscopy. By sequentially acquiring reflection holograms and summing 20 wavefronts equally spaced in spatial frequency in the 485-670 nm range, we are able to achieve a slice-by-slice tomographic reconstruction with a 0.6-1 microm axial resolution in a biological medium. The method is applied to erythrocytes investigation to retrieve the cellular membrane profile in three dimensions.

We present here a three-dimensional evaluation of the amplitude point-spread function (APSF) of a microscope objective (MO), based on a single holographic acquisition of its pupil wavefront. The aberration function is extracted from this pupil measurements and then inserted in a scalar model of diffraction, allowing one to calculate the distribution of the complex wavefront propagated around the focal point. The accuracy of the results is compared with a direct measurement of the APSF with a second holographic system located in the image plane of the MO. Measurements on a 100 x 1.3 NA MO are presented.

Digital holographic microscopy (DHM) is applied to life sciences applications and demonstrate its capability of real-time imaging and quantitative measurements of physiological parameters such as cell volume or mean cell hemoglobin concentration (MCHC) of erythrocyte cells. DHM has the advantage to be non-invasive (no phototoxicity, no contrast agents) and allows a high throughput measurements.

Digital Holographic Microscopy (DHM) is a single shot interferometric technique, which provides quantitative phase images with subwavelength axial accuracy. A short hologram acquisition time (down to microseconds), allows DHM to offer a reduced sensitivity to vibrations, and real time observation is achievable thanks to present performances of personal computers and charge coupled devices (CCDs). Fast dynamic imaging at low-light level involves few photons, requiring proper camera settings (integration time and gain of the CCD; power of the light source) to minimize the influence of shot noise on the hologram when the highest phase accuracy is aimed. With simulated and experimental data, a systematic analysis of the fundamental shot noise influence on phase accuracy in DHM is presented.

We present a procedure that compensates for phase aberrations in digital holographic microscopy by computing a polynomial phase mask directly from the hologram. The phase-mask parameters are computed automatically without knowledge of physical values such as wave vectors, focal lengths, or distances. This method enables one to reconstruct correct and accurate phase distributions, even in the presence of strong and high-order aberrations. Examples of applications are shown for microlens imaging and for compensating for the deformations associated with a tilted thick plate. Finally we show that this method allows compensation for the curvature of the specimen, revealing its surface defects and roughness. Examples of applications are shown for microlenses and metallic sphere imaging.

For what we believe to be the first time, digital holographic microscopy is applied to perform optical diffraction tomography of a pollen grain. Transmission phase images with nanometric axial accuracy are numerically reconstructed from holograms acquired for different orientations of the rotating sample; then the three-dimensional refractive index spatial distribution is computed by inverse radon transform. A precision of 0.01 for the refractive index estimation and a spatial resolution in the micrometer range are demonstrated.

We demonstrate the use of digital holographic microscopy (DHM) as a metrological tool in micro-optics testing. Measurement principles are compared with those performed with Twyman-Green, Mach-Zehnder, and white-light interferometers. Measurements performed on refractive microlenses with reflection DHM are compared with measurements performed with standard interferometers. Key features of DHM such as digital focusing, measurement of shape differences with respect to a perfect model, surface roughness measurements, and optical performance evaluation are discussed. The capability of imaging nonspherical lenses without any modification of the optomechanical setup is a key advantage of DHM compared with conventional measurement tools and is demonstrated on a cylindrical microlens and a square lens array.

This paper presents an optical diffraction tomography technique based on digital holographic microscopy. Quantitative 2-dimensional phase images are acquired for regularly-spaced angular positions of the specimen covering a total angle of pi, allowing to built 3-dimensional quantitative refractive index distributions by an inverse Radon transform. A 20x magnification allows a resolution better than 3 microm in all three dimensions, with accuracy better than 0.01 for the refractive index measurements. This technique is for the first time to our knowledge applied to living specimen (testate amoeba, Protista). Morphometric measurements are extracted from the tomographic reconstructions, showing that the commonly used method for testate amoeba biovolume evaluation leads to systematic under evaluations by about 50%.

The concept of numerical parametric lenses (NPL) is introduced to achieve wavefront reconstruction in digital holography. It is shown that operations usually performed by optical components and described in ray geometrical optics, such as image shifting, magnification, and especially complete aberration compensation (phase aberrations and image distortion), can be mimicked by numerical computation of a NPL. Furthermore, we demonstrate that automatic one-dimensional or two-dimensional fitting procedures allow adjustment of the NPL parameters as expressed in terms of standard or Zernike polynomial coefficients. These coefficients can provide a quantitative evaluation of the aberrations generated by the specimen. Demonstration is given of the reconstruction of the topology of a microlens.

Introducing a microscope objective in an interferometric setup induces a phase curvature on the resulting wavefront. In digital holography, the compensation of this curvature is often done by introducing an identical curvature in the reference arm and the hologram is then processed using a plane wave in the reconstruction. This physical compensation can be avoided, and several numerical methods exist to retrieve phase contrast images in which the microscope curvature is compensated. Usually, a digital array of complex numbers is introduced in the reconstruction process to perform this curvature correction. Different corrections are discussed in terms of their influence on the reconstructed image size and location in space. The results are presented according to two different expressions of the Fresnel transform, the single Fourier transform and convolution approaches, used to propagate the reconstructed wavefront from the hologram plane to the final image plane.

In digital holographic microscopy, shot noise is an intrinsic part of the recording process with the digital camera. We present a study based on simulations and real measurements describing the shot-noise influence in the quality of the reconstructed phase images. Different configurations of the reference wave and the object wave intensities will be discussed, illustrating the detection limit and the coherent amplification of the object wave. The signal-to-noise ratio (SNR) calculation of the reconstructed phase images based on the decision statistical theory is derived from a model for image quality estimation proposed by Wagner and Brown [Phys. Med. Biol. 30, 489 (1985)]. It will be shown that a phase image with a SNR above 10 can be obtained with a mean intensity lower than 10 photons per pixel and per hologram coming from the observed object. Experimental measurements on a glass-chrome probe will be presented to illustrate the main results of the simulations.

We present a method for submicrometer tomographic imaging using multiple wavelengths in digital holographic microscopy. This method is based on the recording, at different wavelengths equally separated in the k domain, in off-axis geometry, of the interference between a reference wave and an object wave reflected by a microscopic specimen and magnified by a microscope objective. A CCD camera records the holograms consecutively, which are then numerically reconstructed following the convolution formulation to obtain each corresponding complex object wavefront. Their relative phases are adjusted to be equal in a given plane of interest and the resulting complex wavefronts are summed. The result of this operation is a constructive addition of complex waves in the selected plane and destructive addition in the others. Tomography is thus obtained by the attenuation of the amplitude out of the plane of interest. Numerical variation of the plane of interest enables one to scan the object in depth. For the presented simulations and experiments, 20 wavelengths are used in the 480-700 nm range. The result is a sectioning of the object in slices 725 nm thick.

We have developed a digital holographic microscope (DHM), in a transmission mode, especially dedicated to the quantitative visualization of phase objects such as living cells. The method is based on an original numerical algorithm presented in detail elsewhere [Cuche et al., Appl. Opt. 38, 6994 (1999)]. DHM images of living cells in culture are shown for what is to our knowledge the first time. They represent the distribution of the optical path length over the cell, which has been measured with subwavelength accuracy. These DHM images are compared with those obtained by use of the widely used phase contrast and Nomarski differential interference contrast techniques.

We present a digital holographic microscope that permits one to image polarization state. This technique results from the coupling of digital holographic microscopy and polarization digital holography. The interference between two orthogonally polarized reference waves and the wave transmitted by a microscopic sample, magnified by a microscope objective, is recorded on a CCD camera. The off-axis geometry permits one to reconstruct separately from this single hologram two wavefronts that are used to image the object-wave Jones vector. We applied this technique to image the birefringence of a bent fiber. To evaluate the precision of the phase-difference measurement, the birefringence induced by internal stress in an optical fiber is measured and compared to the birefringence profile captured by a standard method, which had been developed to obtain high-resolution birefringence profiles of optical fibers.

We present what we believe to be a new digital holographic imaging method that is able to determine simultaneously the distributions of intensity, phase, and polarization state at the surface of a specimen on the basis of a single image acquisition. Two reference waves with orthogonal polarization states interfere with the object wave to create a hologram that is recorded on a CCD camera. Two wave fronts, one for each perpendicular polarization state, are numerically reconstructed in intensity and phase. Combining the intensity and the phase distributions of these two wave fronts permits the determination of all the components of the Jones vector of the object-wave front. We show that this method can be used to image and measure the distribution of the polarization state at the surface of a specimen, and the obtained results indicate that precise quantitative measurements of the polarization state can be achieved. An application of the method to image the birefringence of a stressed polymethyl methacrylate sample is presented.